Telomerase is a large ribonucleoprotein complex responsible for replicating the G-rich strand of telomeres, the physical ends of chromosomes. It is critical for maintaining telomere length and preventing chromosome instability. Telomerase activity is low or undetectable in most somatic cells, while high levels of telomerase activity are essential for viability of most cancer cells. All telomerases contain a large RNA (TR) (451 nt in humans) that includes an RNA template and a unique telomerase reverse transcriptase (TERT), as well as other proteins important for function in vivo. The 5' half of vertebrate TR contains the RNA elements essential for catalysis and TERT binding, while the 3' half contains the elements essential for localization, processing, TR accumulation, and binding the H/ACA RNP proteins. The 5' half of vertebrate TR includes the core domain and the CR4/CR5 domain which, together with TERT, comprise the essential elements of telomerase for catalysis. To address the molecular basis of telomerase catalytic activity, in this grant application we are proposing structural and functional studies of the catalytic core of vertebrate telomerase RNA and its interaction with TERT. Studies will focus on both human telomerase as well as telomerase from medaka fish, a model organism which has the smallest known vertebrate TR. Our overall goals are to determine the global fold and structure of the TR core and CR4/CR5 domains that comprise the catalytic core of telomerase, and provide a view of how they interact with hTERT. We will primarily use NMR methods for structure determination and dynamics, complemented by small angle X-ray scattering (SAXS), electron microscopy (EM), X-ray crystallography, and SHAPE chemistry, to obtain a structural and dynamical view of how telomerase RNA contributes to catalysis and interacts with TERT. Based on the structural data, we will make nucleotide substitutions in the TR to test our hypotheses on the role of the structure in catalysis (nucleotide addition, telomere repeat addition, translocation, processivity, primer binding) and TERT binding. Our specific aims are: (1) Determine the structure and investigate the role of dynamics in the function of the vertebrate TR core domain. The position of the template relative to the pseudoknot will be determined using paramagnetic relaxation enhancement, and dynamics studies will be studied using NMR relaxation measurements and RDCs; (2) Determine the structure and investigate the role of dynamics in the function of the CR4/CR5 domain; and (3) Determine the global fold and interactions between the core and CR4/CR5 domains and hTERT that form the catalytic core of vertebrate telomerase, using a combination of SAXS, EM, and NMR data. These experiments will reveal the structure of the catalytic core of telomerase and the conserved elements important for function. The results of these investigations will provide a molecular basis for understanding telomerase activity as well as how mutations linked to disease affect activity, and for designing drugs that target TR.